1 FIELD OF THE INVENTION . . .
2 BACKGROUND . . .
3 SUMMARY OF THE INVENTION . . .
4 BRIEF DESCRIPTION OF THE DRAWINGS . . .
5 DETAILED DESCRIPTION . . .
5.1 INTRODUCTION . . .
5.2 METHODS FOR DRUG TARGET SCREENING . . .
5.2.1 ALTERNATIVE EMBODIMENTS . . .
5.2.2 APPLICATIONS TO DRUG DISCOVERY . . .
5.3 TRANSCRIPTIONAL STATE EMBODIMENTS . . .
5.3.1 TRANSCRIPT ARRAYS . . .
5.3.2 OTHER METHODS . . .
5.4 MEASUREMENT OF ALTERNATIVE ASPECTS OF BIOLOGICAL STATE . . .
5.5 CELLULAR MODIFICATION METHODS . . .
5.5.1 GENETIC MODIFICATION . . .
5.5.2 OTHER METHODS . . .
5.6 IDENTIFICATION OF GENETIC DRUG TARGETS . . .
6 EXAMPLES . . .
6.1 SYNTHESIS OF LABELED cDNA . . .
6.2 PRODUCTION OF YEAST GENOME MICROARRAYS . . .
6.3 MAKING YEAST DELETION MUTANTS . . .
6.4 PREPARING TRANSCRIPT ARRAY COMPENDIUM . . .
6.5 IDENTIFICATION OF GENETIC TARGET OF A DRUG
6.6 IDENTIFICATION OF CALCINEURIN AS A FK506 TARGET . . .
6.6.1 CYCLOSPORIN AND FK506 . . .
6.6.2 PRODUCTION OF TRANSCRIPT ARRAYS . . .
6.6.3 TARGETS OF CYCLOSPORIN AND FK506 . . .
6.6.4 TARGETS OF CYCLOSPORIN AND FK506 . . .
7 REFERENCES CITED . . .
The field of this invention relates to methods for characterizing the action of drugs in cells, in particular for finding direct targets of drugs, as well as application of these methods to drug discovery.
Drug discovery, a process by which bioactive compounds are identified and preliminarily characterized, is a critical step in the development of treatments for human diseases. Two approaches presently dominate the search for new drugs. The first begins with a screen for compounds that have a desired effect on a cell (e.g., induction of apoptosis), or organism (e.g., inhibition of angiogenesis) as measured in a specific assay. Compounds with the desired activity may then be modified to increase potency, stability, or other properties, and the modified compounds retested in the assay. Thus, a compound that acts as an inhibitor of angiogenesis when tested in a mouse tumor model may be identified, and structurally related compounds synthesized and tested in the same assay. One limitation of this approach is that, often, the mechanism of action and molecular target(s) affected by the compound are unknown, and cannot be determined by the screen. In addition, the assay may provide little information about the specificity of the drug""s effect. Finally, the number of compounds that can be screened by assaying biological effects on cells or animals is limited.
In contrast, the second approach to drug screening involves testing numerous compounds for a specific effect on a known molecular target, typically a cloned gene sequence or an isolated enzyme or protein. For example, high-throughput assays can be developed in which numerous compounds can be tested for the ability to change the level of transcription from a specific promoter or the binding of identified proteins. Although the use of high-throughput screens is an extremely powerful methodology for identifying drug candidates, it has limitations. A major drawback is that the assay provides little or no information about the effects of a compound at the cellular or organismal level. These effects must be tested by using the drug in a series of cell biologic and whole animal studies to determine toxicity or side effects in vivo. In fact, analysis of the specificity and toxicity studies of candidate drugs can consume a significant fraction of the drug development process (see, e.g., Oliff, A and S. H. Friend, xe2x80x9cMolecular Targets for Drug Development,xe2x80x9d in DeVita et al. Cancer: Principles and Practice of Oncoloqy 5th Ed. 1997 Lippincott-Raven Publishers, Philadelphia).
Further, raw data from gene expression analysis are often difficult to coherently interpret. Such measurement technologies typically return numerous genes with altered expression in response to a drug, typically 50-100, possibly up to 1,000 or as few as 10. In the typical case, without more analysis, it is not possible to discern cause and effect from such data alone. The fact that one gene among many has an altered expression in a pair of related biological states yields little or no insight into what caused this change and what the effects of this change are. One is left to ad hoc further experimentation to interpret such gene expression results in terms of biological mechanism. Systematic procedures for guiding the interpretation of such data and such further experimentation, at least in the case of drug target screening, are needed.
Thus, there is a need for improved (e.g., faster and less expensive) methods for characterizing activities and targets of drugs based on effective interpretation of expression data. The present invention provides methods for rapidly characterizing the specificity of candidate drugs and identifying their molecular targets.
The present invention provides methods for identifying targets of a drug in a cell by comparing (i) the effects of the drug on a wild-type cell, (ii) the effects on a wild-type cell of modifications to a putative target of the drug, and (iii) the effects of the drug on a wild-type cell which has had the putative target modified. In various embodiments, the effects on the cell can be determined by measuring gene expression, protein abundances, protein activities, or a combination of such measurements. In various embodiments, modifications to a putative target in the cell can be made by modifications to the genes encoding the target, modification to abundances of RNAs encoding the target, modifications to abundances of target proteins, or modifications to activities of the target proteins. The present invention also provides methods for drug development based on the methods for identifying drug targets.
Accordingly, in a first embodiment, this invention provides a method of determining that a specific cellular constituent present in a cell type is a target of a drug, said method comprising: (a) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that is exposed to said drug in comparison to a cell of said cell type that is not exposed to said drug; (b) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that both is exposed to said drug and also has said specific cellular constituent modified in comparison to a cell of said cell type that has said specific cellular constituent modified and is not exposed to said drug; (c) identifying cellular constituents that drop out by a method comprising determining each of said cellular constituents that is both identified in step (a) as perturbed and that is also identified in step (b) as either differently perturbed or not perturbed; and (d) ascertaining if each said cellular constituent identified in step (c) to drop out is also identified as perturbed in a cell of said cell type that has said specific cellular constituent modified in comparison to a cell of said cell type that does not have said specific cellular constituent modified, whereby said specific cellular constituent is determined as a target of said drug.
In one aspect of the first embodiment, this invention further provides that said ascertaining step further comprises ascertaining if each said cellular constituent that is identified-in step (c) to drop out and is identified as perturbed in said ascertaining step is also identified as similarly perturbed in step (a). In a second aspect of the first embodiment, this invention further provides that step (c) further comprises excluding said specific cellular constituent from said cellular constituents identified to drop out, and wherein step (d) further comprises excluding said specific cellular constituent from said cellular constituents identified as perturbed.
In a second embodiment, this invention provides a method of determining that a specific gene (or genes) or a product of a specific gene (or products of specific genes) present in a cell type is a target of a drug, said method comprising: (a) identifying genes whose expression is perturbed or is not perturbed in a cell of said cell type that is exposed to said drug in comparison to a cell of said cell type that is not exposed to said drug, by a method comprising contacting (e.g., hybridizing) one or more gene transcript arrays with (i) RNA from said cell, or cDNA derived therefrom, exposed to said drug and with (ii) RNA from said cell, or cDNA derived therefrom, not exposed to said drug, wherein said gene transcript array comprises a surface with attached nucleic acids or nucleic acid mimics, said nucleic acids or nucleic acid mimics being capable of hybridizing with RNA species present in said cell type or with cDNA species synthesized from said RNA species; (b) identifying genes whose expression is perturbed or is not perturbed in a cell of said cell type that both is exposed to said drug and also has said specific gene modified in comparison to a cell of said cell type that has said specific gene modified and is not exposed to said drug, by a method comprising contacting one or more gene transcript arrays with (i) RNA from said cell, or cDNA derived therefrom, exposed to said drug and having said specific gene modified and with (ii) RNA from said cell, or cDNA derived therefrom, having said specific gene modified and not exposed to said drug; (c) identifying genes that drop out by a method comprising determining each of said genes that is both identified in step (a) as perturbed and that is also identified in step (b) as either differently perturbed or not perturbed; and (d) ascertaining if each said gene identified in step (c) to drop out is also identified as a gene whose expression is perturbed in a cell of said cell type that has said specific gene modified in comparison to a cell of said cell type that does not have said specific gene modified by a method comprising contacting one or more gene transcript arrays with (i) RNA from said cell, or cDNA derived therefrom, having said specific gene modified and with (ii) RNA from said cell, or cDNA derived therefrom, not having said specific gene modified, whereby said specific gene is determined as a target of said drug.
In one aspect of the second embodiment, this invention further provides that said ascertaining step further comprises ascertaining if each said gene that is identified in step (c) to drop out and is identified as perturbed in said ascertaining step is also identified as similarly perturbed in step (a). In a second aspect of the second embodiment, this invention further provides that step (c) further comprises excluding said specific gene from said genes identified to drop out, and wherein step (d) further comprises excluding said specific gene from said genes identified as perturbed.
In a third embodiment, this invention provides a method of determining one or more drug targets in a cell type comprising: (a) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that is exposed to said drug in comparison to a cell of said cell type that is not exposed to said drug; (b) identifying a specific cellular constituent as a potential drug target if at least one cellular constituent identified in step (a) as perturbed is also identified as similarly perturbed in a cell of said cell type that has said potential drug target modified in comparison to a cell of said cell type that does not have said potential drug target modified; (c) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that both is exposed to said drug and also has said potential drug target modified in comparison to a cell of said cell type that has said potential drug target modified and is not exposed to said drug; (d) identifying cellular constituents that drop out by a method comprising determining each of said cellular constituents that is both identified in step (a) as perturbed and that is also identified in step (c) as either differently perturbed or not perturbed; and (e) ascertaining if each said cellular constituent identified to drop out in step (d) is also identified in step (b) as perturbed, whereby said potential drug target is determined as a drug target.
In one aspect of the third embodiment, this invention further provides for repeating steps (b), (c), (d), and (e) with a different specific cellular constituent modified until all cellular constituents identified in step (a) as perturbed have been identified in step (d) to drop out from modification of at least one of said one or more determined drug targets. In a second aspect of the third embodiment, this invention further provides that perturbation values are identified for said cellular constituents identified as perturbed, and that said ascertaining step further comprises ascertaining, for each cellular constituent identified in step (d) to drop out due to modification of at least two of said one or more determined drug targets, if a combination of perturbation values identified for said cellular constituent in step (b) due to modification of said at least two of said one or more determined drug targets is similar to said perturbation value identified for said cellular constituent in step (a). In a third aspect of the third embodiment, this invention further provides that the combination of perturbation values is preformed by a method comprising adding perturbation values.
In a fourth embodiment, this invention provides a method of determining one or more drug targets in a cell type comprising: (a) performing for each of a plurality of pre-determined cellular constituents, a method comprising identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that has modified a cellular constituent selected from among said plurality of pre-determined cellular constituents in comparison to a cell of said cell type that does not have said selected cellular constituent modified; (b) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that is exposed to said drug in comparison to a cell of said cell type that is not exposed to said drug; (c) determining a specific cellular constituent selected from among said plurality of pre-determined cellular constituents as a potential drug target if at least one cellular constituent identified in step (a) as perturbed when said specific cellular constituent is modified is also identified in step (b) as similarly perturbed; (d) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that both is exposed to said drug and also has said potential drug target modified in comparison to a cell of said cell type that has said potential drug target modified and is not exposed to said drug; (e) identifying cellular constituents that drop out by a method comprising determining each of said cellular constituents that is both identified in step (b) as perturbed and that is also identified in step (d) as either differently perturbed or not perturbed; and (f) ascertaining if each said cellular constituent identified in step (e) to drop out is also identified in step (a) as perturbed when said potential drug target is modified, whereby said potential drug target is determined as a drug target.
In one aspect of the fourth embodiment, this invention further provides that said potential drug target is determined as one specific cellular constituent selected from said plurality of pre-determined cellular constituents for which the greatest number of cellular constituents that are identified in step (a) as perturbed when said specific cellular constituent is modified are also identified in step (b) as similarly perturbed.
In a fifth embodiment, this invention provides a method of determining that a putative drug target is an actual drug target comprising: (a) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that is exposed to said drug in comparison to a cell of said cell type that is not exposed to said drug; (b) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that has said putative drug target modified in comparison to a cell of said cell type that does not have said putative drug target modified; and (c) ascertaining if each said cellular constituent identified as perturbed in step (a) is also identified as perturbed in step (b), whereby said putative drug target is determined as an actual drug target.
In a sixth embodiment, this invention provides a method of determining a more target-specific drug candidate from an initial drug candidate comprising: (a) determining targets of an initial drug candidate by the method of any of the first through the fifth embodiments: (b) modifying the structure of said initial drug candidate; (c) determining targets of said modified initial drug candidate by the method of any of the first through the fifth embodiments; and (d) determining that said modified initial drug candidate is a more target-specific drug candidate than said initial drug candidate if said modified initial drug candidate has fewer targets than said initial drug candidate.
In a seventh embodiment, this invention provides a method of identifying one or more specific cellular constituents present in a cell type that are targets of a drug and that mediate side-effects of the drug, said method comprising: (a) carrying out the method of any of the first through the fifth embodiments for a first drug; (b) carrying out the method of any of the first through the fifth embodiments for a second drug, wherein the first and the second drug are different and exhibit therapeutic efficacy for the same disease or disorder; and (c) identifying those specific cellular constituents determined to be targets of said first drug that are different from those specific cellular constituents determined to be targets of said second drug, thereby identifying one or more specific cellular constituents present in a cell type that are targets of said first drug that mediate side-effects of said first drug.
In an eighth embodiment, this invention provides a method of identifying one or more specific cellular constituents present in a cell type that are targets mediating therapeutic efficacy for a disease or disorder, said method comprising: (a) carrying out the method of any of the first through the fifth embodiments for a first drug; (b) carrying out the method of any of the first through the fifth embodiments for a second drug, wherein the first and the second drug are different and exhibit therapeutic efficacy for the same disease or disorder; and (c) identifying those specific cellular constituents determined to be targets of both said first drug and said second drug, thereby identifying one or more specific cellular constituents present in a cell type that are targets of said first drug that mediate therapeutic efficacy for said disease or disorder.
In a ninth embodiment, this invention provides a method of determining that a specific cellular constituent present in a cell type is a target of a change in the cellular environment, said method comprising: (a) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that is exposed to said change in the cellular environment in comparison to a cell of said cell type that is not exposed to said change in the cellular environment; (b) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that both is exposed to said change in the cellular environment and also has said specific cellular constituent modified in comparison to a cell of said cell type that has said specific cellular constituent modified and is not exposed to said change in the cellular environment; (c) identifying cellular constituents that drop out by a method comprising determining each of said cellular constituents that is both identified in step (a) as perturbed and that is also identified in step (b) as either differently perturbed or not perturbed; and (d) ascertaining if each said cellular constituent identified in step (c) to drop out is also identified as perturbed in a cell of said cell type that has said specific cellular constituent modified in comparison to a cell of said cell type that does not have said specific cellular constituent modified, whereby said specific cellular constituent is determined as a target of said change in the cellular environment.